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In response to an intracellular infectious agent, the immune system produces a specific cellular response as well as a T cell-dependent Ab response. Precursor T cells differentiate into effector T cells, including Th1 cells, and T follicular helper (TFH) cells. The latter cooperate with B cells to form germinal centers and induce the formation of Ab-forming plasmacytes. One major focal point for control of T cell differentiation is the transcription factor BCL6. In this study, we demonstrated that the Bcl6 gene is regulated by FOXO1-binding, cis-acting sequences located in a highly conserved region of the first Bcl6 intron. In both mouse and human T cells, deletion of the tandem FOXO1 binding sites increased the expression of BCL6 and enhanced the proportion of TFH cells. These results reveal a fundamental control point for cellular versus humoral immunity.

BACKGROUND There is an unmet need for developing faithful animal models for preclinical evaluation of immunotherapy. The current approach to generate preclinical models for immunotherapy evaluation has been to transplant CD34+ cells from umbilical cord blood into immune-deficient mice followed by implantation of patient derived tumor cells. However, current models are associated with high tumor rejection rate secondary to the allograft vs. tumor response from human leukocyte antigen (HLA) mismatches. We herein report the first development of a novel, humanized patient-derived xenograft (PDX) model using autologous CD34+ cells from bone marrow aspirate obtained from a patient with metastatic clear cell renal cell carcinoma (mRCC) from whom a PDX had been developed. CASE DESCRIPTION This is a 68-year-old Caucasian man diagnosed with mRCC with metastasis to the liver in 2014. He was treated with sunitinib +/- AGS-003 and underwent a cytoreductive right nephrectomy, left adrenalectomy and partial liver resection. PDX was generated using resected nephrectomy specimen. After surgery, patient received multiple lines of standard of care therapy including sunitinib, axitinib, bevacizumab, everolimus and cabozantinib. While progressing on cabozantinib, he was treated with nivolumab. Seven years after initiation of nivolumab, and 4 years after stopping systemic therapy, he remains in complete remission. To generate autologous PDX model, bone marrow aspirate was performed and CD34+ hematopoietic stem/progenitor cells (HSPCs) were isolated and injected into 150 rad irradiated non-obese diabetic scid gamma null (NSG) mice. At 11 weeks post-transplant, the matched patient PDX was injected subcutaneously into the humanized mice and the mice were treated with nivolumab. CONCLUSIONS Our case represents successful therapy of nivolumab in mRCC. Furthermore, HPSCs obtained from a single bone marrow aspirate were able to reconstitute an immune system in the mice that allowed nivolumab to inhibit the tumor growth of PDX and recapitulated the durable remission observed in the patient with nivolumab. We observed the reconstitution of human T cells, B cells and natural killer (NK) cells and unlike the humanized mouse model using cord blood, our model system eliminates the tumor rejection from mis-matched HLA. Our autologous humanized renal cell carcinoma (RCC) PDX model provides an effective tool to study immunotherapy in a preclinical setting.

Imaging strategies to monitor chimeric antigen receptor (CAR) T-cell biodistribution and proliferation harbor the potential to facilitate clinical translation for the treatment of both liquid and solid tumors. In addition, the potential adverse effects of CAR T cells highlight the need for mechanisms to modulate CAR T-cell activity. The herpes simplex virus type 1 thymidine kinase (HSV1-tk) gene has previously been translated as a PET reporter gene for imaging of T-cell trafficking in patients with brain tumor. The HSV1-TK enzyme can act as a suicide gene of transduced cells through treatment with the prodrug ganciclovir. Here we report the molecular engineering, imaging, and ganciclovir-mediated destruction of B7H3 CAR T cells incorporating a mutated version of the HSV1-tk gene (sr39tk) with improved enzymatic activity for ganciclovir. The sr39tk gene did not affect B7H3 CAR T-cell functionality and in vitro and in vivo studies in osteosarcoma models showed no significant effect on B7H3 CAR T-cell antitumor activity. PET/CT imaging with 9-(4-[18F]-fluoro-3-[hydroxymethyl]butyl)guanine ([18F]FHBG) of B7H3-sr39tk CAR T cells in an orthotopic model of osteosarcoma revealed tumor homing and systemic immune expansion. Bioluminescence and PET imaging of B7H3-sr39tk CAR T cells confirmed complete tumor ablation with intraperitoneal ganciclovir administration. This imaging and suicide ablation system can provide insight into CAR T-cell migration and proliferation during clinical trials while serving as a suicide switch to limit potential toxicities. SIGNIFICANCE: This study showcases the only genetically engineered system capable of serving the dual role both as an effective PET imaging reporter and as a suicide switch for CAR T cells.